The goal of these studies is to understand the interactions of signaling proteins associated with plasma-lemmal caveolae in vascular endothelial cells, with an ultimate aim of identifying the abnormalities in endothelial signaling that underlie many vascular disease states. Caveolae have a distinctive lipid content, and serve as sites for the sequestration of diverse signaling molecules, including receptors as well as protein and lipid kinases. The protein caveolin serves both structural and signaling roles in caveolae, and modulates interactions between the plasmalemma and cytoskeleton. The proposed studies will use cellular imaging and biochemical approaches to explore the role of caveolin in modulating protein interactions that couple receptor activation to signaling responses. In the previous project period, we developed siRNA-based approaches to "knock-down" expression of caveolin and other key signaling proteins, and explored the consequences for cellular responses in endothelial cells. We applied fluorescence resonance energy transfer (FRET) imaging methods to characterize spatial and temporal features of the interactions between calmodulin and eNOS. We discovered potentially important signaling roles for caveolin in regulation of cytoskeleton-associated signaling proteins such as Rac1, VASP and MARCKS, and identified pathways for crosstalk between the platelet-derived lipid sphingosine 1-phosphate (S1P) and VEGF signaling. The proposed studies will apply siRNA-based methods to explore roles for caveolin in the regulation of cytoskeleton-associated signaling proteins. We will exploit FRET methods to explore caveolin-modulated interactions between calmodulin, MARCKS, and eNOS, and will determine the effects of calmodulin phosphorylation on its protein interactions. We will identify the roles of caveolin and MARCKS in regulation of phosphoinositide targeting to caveolae. We will determine the effects of caveolin on SIP1 receptor targeting and turnover, and will explore the effects of caveolin on S1P-mediated calcium mobilization and kinase activation. The proposed studies will identify mechanisms whereby caveolin and caveolae- associated proteins regulate agonist-mediated cellular processes such as angiogenesis and endothelial permeability. Our proposed studies of cellular and molecular mechanisms that regulate protein interactions in cardiovascular signaling may lead to the identification of new points for pharmacological intervention.